Killer Knots: Molecular evolution of Inhibitor Cystine Knot toxins in wandering spiders (Araneae: Ctenidae)

Venom expressed by the nearly 50,000 species of spiders on Earth largely remains an untapped reservoir of a diverse array of biomolecules with potential for pharmacological and agricultural applications. A large fraction of the noxious components of spider venoms are a functionally diverse family of structurally related polypeptides with an inhibitor cystine knot (ICK) motif. The cysteine-rich nature of these toxins makes structural elucidation difficult, and most studies have focused on venom components from the small handful of medically relevant spider species such as the highly aggressive Brazilian wandering spider Phoneutria nigriventer. To alleviate difficulties associated with the study of ICK toxins in spiders, we devised a comprehensive approach to explore the evolutionary patterns that have shaped ICK functional diversification using venom gland transcriptomes and proteomes from phylogenetically distinct lineages of wandering spiders and their close relatives. We identified 626 unique ICK toxins belonging to seven topological elaborations. Phylogenetic tests of episodic diversification revealed distinct regions between cysteine residues that demonstrated differential evidence of positive or negative selection, which may have structural implications towards the specificity and efficacy of these toxins. Increased taxon sampling and whole genome sequencing will provide invaluable insights to further understand the evolutionary processes that have given rise to this diverse class of toxins.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy

Immunomodulation has been considered an important approach in the treatment of malignant tumors. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumor size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated twenty-three subfractions named Low Weight (LW-1 to LW-12) and High Weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumor cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

EXTH-42. SPIDER VENOM COMPONENTS DECREASE GLIOBLASTOMA CELL MIGRATION AND INVASION THROUGH RHOA-ROCK AND NA+/K+-ATPASE β2: POTENTIAL MOLECULAR ENTITIES TO TREAT INVASIVE BRAIN CANCER

Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells. The involvement of RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) was also evaluated. Human (NG97) GB cells were treated with twelve subfractions (SFs - obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound heling and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Two (SF1 and SF11) of twelve SFs decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2. All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway. Support: FAPESP #2015/04194-0; CNPq #431465/2016–9.

DDRE-19. SPIDER VENOMS AS SOURCES OF MOLECULES FOR NEW CANCER TREATMENTS: THE EFFECTS OF THE SNX-482 PEPTIDE ON MACROPHAGE MODULATION

Malignant primary brain tumors remain among the most difficult cancers to treat. In malignant tissues, macrophages are accumulated in a high infiltration being known as tumor-associated macrophages (TAMs). These cells are associated with poor prognostics in many types of cancer. Studies in our group demonstrated that the venom of Phoneutria nigriventer (PnV), a wandering spider from South America, has reduced the development of glioblastoma (GBM) in a murine model, inducing a large infiltrate of TAMs. Subsequently, in vitro results demonstrated that PnV activates macrophages, increasing the ability to kill tumor cells. The aim of this study was to analyze the effects of the peptide SNX-482 presented in the venom of Hysterocrates gigas in macrophages for further correlation with the PnV. Macrophages were differentiated from bone marrow precursors collected from male C57BL6 mice and differentiated for 7 days with M-CSF. These cells were used for polarization and coculture with T cells and analyzed by flow cytometry. PCR Array was also performed (QIAGEN) for the analysis of gene expression. The results showed that SNX-482 could activate macrophages in a not dose-dependent response. There was an increase in the main activation markers (CD40, CD80, CD86, CD68, CD83, and MHCII). The polarization indicated that the peptide potentiated the proinflammatory effect of M1 macrophages (increased MHCII and iNOS). The screening of 86 cancer-related genes showed that the Ccr4, Pdcd1, Gzmb, and IFN-γ genes had an increase in their expression. Furthermore, we developed in C57BL/6 mouse a pre-clinical model of intracranial glioblastoma using the Gl261 cell line. The results showed an applied-easy-safe model that could alter the gene expression of cancer markers. Taken together, all the results contributed to increasing the knowledge about the peptide SNX-482 and the model for further pre-clinical assays of glioblastoma, making a great advance in the development of new treatments.

EXTH-28. MOLECULAR CHARACTERIZATION OF HUMAN GLIOMAS AND IDENTIFICATION OF NEOPLASMS RESPONSIBLE FOR NEW BIOPHARMACEUTICALS OBTAINED FROM ANIMAL VENOM: A TRANSLATIONAL APPROACH

Gliomas correspond to approximately 80% of primary malignant brain tumors in adults. Associate histopathological classification to the identification of the molecular profile of these tumors is a great strategy to predict the tumor’s behavior and its responsiveness to the treatments. Studies have shown the potential of using biopharmaceuticals against cancer. Our research group has recently found two main subfractions isolated from Phoneutria nigriventer spider venom (PnV) (called SF1 and SF11) that can act decreasing the number of migrating cells during transwell assay. Therefore, the present study aimed to characterize, as regards molecular identity, the responsiveness of glioma samples collected from patients to the molecules purified from the PnV, in order to improve diagnostic and prognostic predictions, as well as contribute to the development of new drugs. After consent of the patients, samples of the tumors clinically diagnosed as glioma were collected during the surgical procedure to cultivation, that were performed until 10 passages to establish a lineage before use. To analyze its responsiveness to PnV in a migration assay, cultured human glioblastoma (1-JA63) cells were treated with PnV-isolated (HPLC) subfractions (SF1 and SF11 at 1 µg/ml) for 12 and 48 h (control stayed in IMDM) in a transwell system. SFs (mainly SF1) treatment induced a significant reduction in the number of migrating cells after 48 hours of exposure, in comparison to the control (untreated). Ongoing trials continue the establishment of another tumor samples of gliomas and migration tests with PnV subfractions will be carried out, to compare their effects in relation to other degrees of the disease also correlating the tumor response to the drug with the molecular profile. The results will contribute to the development of a potential individualized therapy also adding information about the correlation between the molecular identity of the tumor and its prognosis.